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Updated: Nov 4, 2025
Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
Published on: May 23, 2025
High-throughput CRISPR-mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance
Taraka S P Grandhi1, Jeremy To, Angelica Romero
1Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.
Abstract:
Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.
Insights
Cancer cells evolve resistance to drugs through mutations. A new 3D CRISPR screening platform rapidly identifies these resistance mutations, improving drug discovery and treatment strategies for cancer patients.
Area of Science:
- Oncology
- Genomics
- Drug Discovery
Background:
- Cancer is driven by somatic mutations, leading to tumor evolution and drug resistance.
- Current therapeutic strategies face challenges from molecular evolution of cancer cells.
- There is a need for advanced platforms to discover drug resistance mechanisms.
Purpose of the Study:
- To develop and validate a pooled in vitro CRISPR screening platform for rapid discovery of drug resistance mutations.
- To identify genes conferring resistance to CDK4/6 inhibitors (CDK4/6i) in a 3D cancer model.
- To compare the efficacy of 3D versus 2D screening platforms for identifying resistance mechanisms.
Main Methods:
- Utilized a pooled in vitro CRISPR screening platform in a 3D multicellular tumor spheroid (MCTS) model.
- Exposed gene-edited MCTS to CDK4/6 inhibitors to select for drug-resistant phenotypes.
- Employed next-generation sequencing to detect enriched drug-resistant mutations after 28 days.
- Performed genome-wide CRISPR-mediated knockout screen (>18,000 genes).
Main Results:
- The 3D CRISPR screening platform rapidly identified mutations conferring resistance to CDK4/6 inhibitors.
- The platform demonstrated sensitivity in enriching for rare drug-resistant cells within complex 3D spheroids.
- Genome-wide screening identified known and novel candidate genes associated with CDK4/6i resistance.
- Several novel resistance genes were exclusively identified in the 3D assay, not in conventional 2D assays.
Conclusions:
- The 3D CRISPR screening platform enables rapid, unbiased discovery of drug resistance mutations in a complex tumor microenvironment.
- This platform can identify novel resistance mechanisms and improve the reconfirmation of known ones.
- Incorporating 3D resistance profiling may enhance decision-making in drug discovery and treatment strategies.
- The platform facilitates the study of non-cell autonomous interactions and clonal competition in drug resistance.
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